The objective of this work is to understand the physiological role of enzymatic protein carboxyl methylation reactions in human tissues and other cells. We propose to continue our studies of an L-isoaspartyl/D-aspartyl methyltransferase (E.C. 2.1.1.77) that catalyzes the modification of damaged proteins containing these unusual residues. This enzyme is found in the cytosolic fraction of all mammalian tissues examined and recognizes the altered residues that result from spontaneous racemization, isomerization, and deamidation of normal L-aspartyl and L-asparaginyl residues in aged proteins. In model systems, the formation of methyl esters at L-isoaspartyl residues can lead to their conversion to L-aspartyl residues and suggests that this enzyme functions to repair certain types of covalent damage to intracellular proteins and limit their accumulation in aging cells. We will use a combination of biochemical and molecular biological techniques in in vitro and in intact cell systems, focusing particularly on human erythrocytes. Since the structure of this enzyme has been remarkably well conserved from bacteria to human cells, we will also utilize genetically-manipulatable microbial model systems. This methyltransferase may represent one of the first members of a class of enzymes that can check spontaneous damage to cellular proteins, and its disruption in pathological conditions may both decrease their useful lifetime and contribute to accelerated aging processes. our recent discovery of a new class of protein carboxyl methyltransferases suggests that these enzymes may also have roles in the regulation of cellular signalling reactions. Proteins that contain a C-terminal tetrapeptide sequence are modified by reactions that form C-terminal S-isoprenylated cysteine methyl esters. These proteins include the ras oncogene products as well as other small G-proteins, subunits of the large G-proteins, and nuclear lamin components. The isoprenylation and methylation of these proteins have been postulated to lead to both membrane association and to the control of their signalling activities. We will purify and characterize the enzymes involved in these reactions from both mammalian tissues and yeast. The ability to pharmacologically control these modification enzymes, and thus signalling proteins involved in cell division, may represent new therapies for cancer and other diseases.